TABLE 4.
Kinetic parameters for wild-type CCT236 and active site mutants of CCT236
Parameters were obtained from the plots of initial velocity versus substrate concentration shown in Fig. 6, using GraphPad Prism 4 software. The Km and Vmax values are derived from the best fit to the Hill equation (see Fig. 6, legend) ± S.E. of the fit. The wild-type Vmax value from the CTP curve is more accurate, because the CTP concentration was subsaturating in the phosphocholine curves. Units for Vmax are nanomoles of CDP-choline formed (min−1) (mg CCT−1). Units for K* are in millimolar.
| CCT construct | CTP dependence |
Phosphocholine dependence |
||||
|---|---|---|---|---|---|---|
| Vmax | K* | Hill coefficient | Vmax | K* | Hill coefficient | |
| CCT236 wild type | 3399 ± 365 | 7.3 ± 1.1 | 1.6 ± 0.3 | 1724 ± 108 | 0.33 ± 0.09 | 1.4 ± 0.2 |
| Y173A | 167 ± 21 | 7.7 ± 1.2 | 1.9 ± 0.3 | 116 ± 11 | 0.48 ± 0.15 | 1.4 ± 0.2 |
| H168A | 34 ± 3 | 7.7 ± 1.0 | 2.0 ± 0.3 | 69 ± 12 | 2.5 ± 0.6 | 1.3 ± 0.3 |
| H168A,Y173A | 21 ± 6 | 8.8 ± 2.9 | 2.0 ± 0.6 | 55 ± 16 | 3.2 ± 1.2 | 1.4 ± 0.4 |